FDA-approved PDE4 inhibitors alleviate the dominant toxicity of ALS–FTD-associated CHCHD10S59L by reducing the PINK1/Parkin pathway

Background Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) have been identified as a genetic cause of amyotrophic lateral sclerosis and/or frontotemporal dementia(ALS-FTD). In our previous studies using in vivo Drosophila model expressing CHCHD10S59L, and human cell models expressing CHCHD10S59L, we have identified that the PINK1/Parkin pathway is activated and causes cellular toxicity. Furthermore, we demonstrated that pseudo-substrate inhibitors for PINK1 and mitofusin2 agonists mitigated the cellular toxicity of CHCHD10S59L. Evidences using in vitro, in vivo genetic, and chemical tools indicate that inhibiting PINK1 would be the most promising treatment for CHCHD10S59L-induced diseases. Methods An in vivo human cell culture and in vivo Drosophila models expressing CHCHD10S59L mutant were utilized in this study to evaluate the effect of PDE4 inhibitors in PINK-parkin mediated cytotoxicity through immunohistochemical and seahorse assays. Data were analysed using one-way ANOVA and post-hoc Dunnett’s test for statistical significance. Results We investigated cellular pathways that can modulate the PINK1/Parkin pathway and reduce CHCHD10S59L-induced cytotoxicity. Here, we report that FDA-approved PDE4 inhibitors reduced CHCHD10S59L-induced morphological and functional mitochondrial defects in human cells and an in vivo Drosophila model expressing C2C10HS81L. Multiple PDE4 inhibitors decreased PINK1 accumulation and downstream mitophagy induced by CHCHD10S59L. Conclusion These findings suggest that PDE4 inhibitors currently available in the market may be repositioned to treat CHCHD10S59L-induced ALS-FTD and possibly other related diseases, and that disease treatment with PDE4 inhibitors should include careful consideration of the PINK1/Parkin pathway, as it is generally recognized as a protective pathway.

We reported that CHCHD10 S59L overexpression in human cells induced fragmented mitochondria and mitochondrial dysfunction.Consistently, expressing C2C10H S81L (Drosophila homolog of human CHCHD2 and CHCHD10 bearing a mutation in the conserved postion with human S59L) with the tissuespeci c GAL4/UAS expression system, induced rough eye phenotypes, neuromuscular junction defects, and muscle degeneration with fragmented mitochondria (2).Through a series of genetic interaction studies, we identi ed that PINK1 and Parkin are strong modi ers for the dominant toxicity of C2C10H S81L .RNAi-mediated knockdown of PINK1 and Parkin strongly suppressed the rough eye phenotypes, muscle degeneration, and mitochondrial fragmentation of C2C10H S81L .Additionally, ATP production was recovered in the adult indirect ight muscles.In contrast, overexpression of PINK1 and Parkin signi cantly enhanced the rough eye phenotypes (2).CHCHD10 S59L expression in human cells including HeLa and SH-SY5Y induced mitochondrial accumulation of PINK1 and Parkin, and subsequently, LC3 which is a marker for activated mitophagy (2).siRNA transfection for PINK1 and Parkin reduced mitochondrial fragmentation induced by CHCHD10 S59L   and also improved functional mitochondrial respiration signi cantly.When we knocked-down mitophagy adaptors for the PINK1/Parkin pathway, it showed recovery in functional mitochondrial respiration in multiple cell lines.Interestingly, CHCHD10 S59L induced mitochondrial fragmentation and dysfunction in HeLa that does not express Parkin.siRNA mediated knock-down of PINK1 reversed the mitochondrial network fragmentation in two patient-derived broblasts (2).Therefore, these results indicate that reducing PINK1 expression or activity can be a potential therapeutic strategy.
In fact, we identi ed two psudosubstrate inhibitors for PINK1 and demonstrated that two peptide inhibitors reduced its ubiquitin kinase activity and mitigated CHCHD10 S59L -induced mitochondria fragmentation (2).However, due to the lack of small molecule PINK1 inhibitors, we could not evaluate the e cacy of PINK1 inhibition in vivo.Therefore, we searched small molelcules that may reduce PINK1 expression level or enzymatic activity directly or indirectly.
In 2016, Akabane et al. reported that PKA regulates the stability of PINK1 via phosphorylating mito lin (3).In that study, they demonstrated that Parkin recruitment to depolarized mitochondria was severely inhibited by Forskolin treatment via increase in cAMP levels and activation of a protein kinase A (PKA).Activation of PKA and subsequent phosphorylation of MIC60 (which is a substrate of PKA) impaired the translocation of Parkin to damaged mitochondria via reduced PINK1 protein stability.Therefore, we evaluated the e cacy of PDE4 inhibitors for ameliorating CHCHD10 S59L -induced toxicity in human cells and Drosophila.Here, we report that FDA-approved PDE4 inhibitors mitigate CHCHD10 S59 L (C2C10H S81L )-induced toxicity in human cells and Drosophila by reducing the PINK1/Parkin pathway.

DNA constructs.
All cDNAs for human wild-type and mutant forms of CHCHD10 were synthesized and inserted in the pcDNA3 vector containing FLAG-tag by Genescripts and mCherryLC3B (#40827) plasmid was obtained from Addgene.
Cell culture and transfections.HeLa cells, HeLa Parkin−YFP , HeLa PINK1−V5 cells were gifts from Dr. Richard Youle's lab.Cells were maintained in Dulbecco-modi ed Eagle medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 1× penicillin/streptomycin (Invitrogen), and 1 x GlutaMax (Gibco).Cells were transfected using jetPRIME (Polyplus #89129-924) transfection reagent according to the manufacturer's protocol.For plasmid transfections, around 3 X10 4 cells were counted using ADAM-MC2 cell counter (NanoEntek) and plated in a 4-well chambered slides in a drug containing media and allowed to grow for 24 hrs.Transfections were carried out with 0.5 ug of plasmid and 1ul of jetPRIME transfection reagent (1:2) mixed in 50uL of jetPRIME buffer per well.In cases of co-transfections, plasmids were mixed at equimolar concentrations.Media was changed with a fresh drug containing media after 4 hrs and incubated with Mito-RFP (CellLight™ Mitochondria-RFP, BacMam 2.0) for overnight.After 24 hrs of the transfection, cells were xed and processed for immnuostaining.
Immuno uorescence and antibodies.
Cells were xed with 4% paraformaldehyde in 1XPBS for 10 mins, permeabilized with 0.1% Triton X-100 for 10 mins and blocked with 5% BSA in PBS for 1 hr at room temperature.Primary antibodies were diluted in 5% BSA in 1XPBS and slides were incubated overnight at 4°C.Slides were then rinsed thrice with 1XPBST(0.1% tween-20) and incubated with secondary antibodies for 1.5 hrs at room temperature.
Slides were washed again and mounted with coverglass using Prolong Diamond Antifade Reagent with DAPI (Invitrogen; P36962).Images were obtained with a LSM 710 (Zeiss, X63, 1. Mitochondrial respiratory activity assay. Mitochondrial respiration in HeLa, HeLa Parkin−YFP cells were measured using the Seahorse Extracellular Flux Analyzer XFp (Agilent Technologies, #102416-100) with XF Cell Mito Stress Test Kit (Agilent Technologies, #103015-100).Approximately, 1 x 10 4 transfected cells were plated into desired drugcontaining media in V3-PS 96 well plate the day before performing an assay.Next day, media were replaced with fresh assay media supplemented with 1 mM pyruvate, 2 mM glutamine and 15 mM glucose with a pH adjusted to 7.6.The standard mitochondria stress tests were performed consisting of basal measurements followed by measurements after sequential addition of 1 uM oligomycin, 0.5 uM FCCP and 0.5 uM Rotenone/antimycin A. At the end of the assay, protein concentration of each well were determined using BCA assay to normalize the obtained OCR values.

Generation of Drosophila lines.
Transgenic Drosophila lines were generated by BestGene with the standard PhiC31 integrase-mediated transgenesis system.All transgenes were inserted into the third chromosome attp2 site to avoid positional gene expression differences.Fly cultures and crosses were performed on standard y food (Genesee Scienti c) and raised at 25°C with a 12:12 hr light: dark cycle.MHC-GAL4 was used as driver for the expression in the muscles.
Drosophila adult muscle preparation and immunohistochemistry.
The sarcomere structure and mitochondrial morphology of the indirect ight muscle were analyzed as previously described(2) Adult ies were xed with 4% paraformaldehyde in 1X PBS for 1 hour after a quick dissection,then immersed in OCT compound (Fisher Scienti c) and ash frozen in liquid nitrogen or dry ice.Fixed tissues (~ 15-16 microns) were sectioned by a cryomicrotome (Leica) and directly mounted on the slide.Additional post xing was performed with 4% paraformaldehyde in 1XPBS for 10 mins at room temperature and then permeabilized with 0.2% Triton X-100 in 1XPBS and blocked with 5% BSA solution for 1 hr.Sections were incubated with phalloidin-Alexa Fluor 594 (Invitrogen) overnight at 4°C for muscle staining and mounted with Prolong Diamond Antifade Reagent with DAPI.Images were obtained with LSM 710 confocal microscope (Carl Zeiss) with 63X magni cation.

Drosophila ATP assay.
Fly thoraces were dissected out, collected and homogenized in 20 µl of homogenization buffer (100 mM Tris, 4 mM EDTA and 6 M guanidine-HCL, pH 7.8).The homogenate was centrifuged at 16,000 x g for 10 min and the supernatant was collected and diluted to 1:200 with deiodinized water for ATP measurement using the Cell Titer-Glo luminescent cell viability assay kit (Promega, G7571) and 1:10 for determining the protein concentration.Data was obtained by normalizing the ATP concentration with protein amount.
Image analysis and statistical analysis.
Mitochondrial length was measured using MiNA tool set combined with ImageJ software as previously described (21).Statistical analysis was performed with Prism5(GraphPad) software.Statistical signi cance is expressed as p value that was determined with student's t-test, one-way or two-way analysis of variance.One-or two-way ANOVA was followed by the indicated post-hoc tests.1A-B).As expected, we also successfully detected that Forskolin treatment blocked PINK1 accumulation signi cantly (Fig. 1C-D).

Forskolin blocks PINK1 and Parkin accumulation
These observations led us to examine the effects of Forskolin on CHCHD10 S59L -induced mitochondrial toxicity.
Forskolin and Ro umilast mitigate the toxicity of CHCHD10 S59L in cell models.
When we treated Forskolin to HeLa cells transiently transfected with CHCHD10 S59L , mitochondrial branch length increased signi cantly compared to that of DMSO-treated cells (Fig. 1E).Forskolin did not increase mitochondrial branch length in CHCHD10 WT or empty vector transfected groups(Fig.1E).Since Forskolin increases cyclic AMP levels by activating adenylate cyclases, we investigated whether phosphodiesterase inhibitors (PDE inhibitors) could induce the same effect by preventing cAMP degradation.First, we chose Ro umilast, a selective PDE4 inhibitor, that was approved and marketed for chronic obstructive pulmonary disease (COPD)(4) (5).When we treated Ro umilast (1 µM or 10 µM for 24hrs) to CHCHD10 S59L -expressing HeLa cells, mitochondrial fragmentation caused by CHCHD10 S59L was signi cantly reduced while aggregation of CHCHD10 S59L was not affected (Fig. 1F,G,H).
We previously reported that CHCHD10 S59L induced PINK1 accumulation on mitochondria and reduction of PINK1 expression or activity was bene cial to mitochondria morphologically and functionally (2).Therefore, we examined whether Forskolin and Ro umilast reduced CHCHD10 S59L -induced PINK1 acculumation in HeLa cells.For this purpose, we used HeLa cells stably expressing V5 tagged PINK1 (HeLa PINK1−V5 cells)(6).When CHCHD10 S59L was transfected and expressed, more than 80% of cells showed PINK1 accumulation on mitochondria and the PINK1 aggregation-positive cells were signi cantly decreased by Forskolin and Ro umilast treatment (Fig. 2A-B).Acute treatment of 50 µM Forskolin and 24 hour-treatment of 20 µM Forskolin showed similar effects (Fig. 2B).However, we did not observe signi cant dose-dependent effects of Ro umilast when it was treated with 1 µM or 10 µM concentrations in this experiment (Fig. 2B).When CHCHD10 S59L was transfected and expressed, about 70% of cells had Parkin-YFP punctae colocalized with mitochondria (Fig. 2C).Although acute 50 µM Forskolin treatment reduced Parkin-YFP accumulation, 24 hour-treatment of 20 µM Forskolin treatment showed a more signi cant decrease of Parkin accumulation on mitochondria (Fig. 2D).Interestingly, a relatively low dose of Ro umilast (1 µM) signi cantly reduced Parkin-YFP accumulation more than 10 µM Ro umilast (Fig. 2D).This may be due to the dynamic regulation of cytosolic cAMP levels by cAMP-activated PKA and PKA-activated phosphodiesterase (7).Consistently, the seahorse analysis revealed that Forskolin and/or Ro umilast treatment increased basal mitochondrial respiration and ATP production with increased maximal respiration (Fig. 2E).The spare capacity was also increased minimally and not signi cantly in Ro umilast treated samples (Fig. 2E).The reduction of Parkin accumulation was also observed in a neuroblastoma cell line, SH-SY5Y, with transiently transfected mCherry-Parkin (Suppl Fig S1 A-B).
Forskolin and Ro umilast reduced LC3 accumulation on mitochondria.
Forskolin and Ro umilast ameliorated mitochondrial defects induced by C2C10H S81L in Drosophila.
Finally, we investigated the effect of Forskolin and Ro umilast treatment on mitochondrial defects in Drosophila expressing C2C10H S81L .For this, we expressed C2C10H S81L in muscle tissues with the MHC-GAL4 driver.The C2C10H S81L ies were reared in y food containing Forskolin (50 uM), Ro umilast (40 uM), and DMSO.Indirect ight muscle sections of C2C10H S81L ies stained with a uorophore conjugated streptavidin (green) and phalloidin (red) revealed fragmented mitochondria along with muscular degeneration (Fig. 3C, DMSO).This condition was ameliorated signi cantly in Forskolin and Ro umilast treated ies (Fig. 3C).ATP levels were measured as an indicator of mitochondrial dysfunction in the y thoraxes which primarily consists of muscle tissues.We observed signi cantly improved ATP levels in the muscle tissues of ies expressing C2C10H S81L treated with Forskolin and Ro umilast, as compared to DMSO-fed C2C10H S81L ies (Fig. 3D).Therefore, Forskolin and Ro umilast imparted a rescue effect on the mitochondrial defects induced by toxic C2C10H S81L .Other PDE4 inhibitors, Apremilast and Rolipram, are also effective.
To further validate the potential of PDE4 inhibitors against CHCHD10 S59L mediated toxicity, we also tested the rst generation PDE4 inhibitor, Rolipram (9,10), and another approved drug, Apremilast, which is prescribed for plaque psoriasis or psoriatic arthritis (9,11,12).Both Apremilast and Rolipram reduced Parkin-YFP accumulation in HeLa YFP−Parkin cells transiently transfected with CHCHD10 S59L (Fig. 4A). 1 µM Apremilast reduced Parkin-YFP accumulation to a comparable level with 20µM Ro umilast.However, Rolipram only showed marginal e cacy though it was statistically signi cant in multiple independently repeated experiments (Fig. 4B).The seahorse analysis also veri ed that both Apremilast and Rolipram reduced the toxicity of CHCHD10 S59L while Apremilast was more effective than Rolipram in rescuing defective mitochondrial function caused by CHCHD10 S59L (Fig. 4C, S2 A-C).A similar trend in mitochondrial respiration was also re ected in the seahorse assay using SHSY5Y neuroblastoma cells (Fig S1 C).

DISCUSSION
Mitochondrial dysfunction remains one of the prominent pathological features of neurodegenartive disorders including ALS-FTD (13).Emerging reports suggested that cAMP signaling plays a key role in mitochondrial biology and homeostasis (14).Baek and colleagues demonstrated that CHCHD10 S59L mutant induced dominant toxicity in Drosophila and HeLa cells, and the PINK1/Parkin mediated pathway was implicated in CHCHD10 associated ALS-FTD (2).Here, we demonstrate that FDA-approved PDE4 inhibitors successfully reduced morphological and functional mitochondrial defects in human cell lines and in vivo Drosophila model expressing CHCHD10 S59L and C2C10H S81L , respectively.
We observed that Forskolin treatment reduced the Parkin and PINK1 accumulation in HeLa YFP−PARKIN and HeLa PINK1−V5 cells, respectively, after depolarization by an uncoupling gent as previously reported by Akabane and colleagues (3).Forskolin, being an adenylate cyclase activator, increased the cAMP levels of the system and activated the PKA pathway and led to destabilization of PINK1 on depolarized mitochondria.Forskolin also reduced the disrupted mitochondrial network and enhanced mitochondrial respiration in transiently transfected CHCHD10 S59L HeLa cells.Similarly, Ro umilast treatment to CHCHD10 S59L expressing cells exhibited a rescued phenotype.Both drugs act through the same mechanism i.e via cAMP/PKA pathway by increasing the cAMP levels in the cellular pool, one by hydrolyzing ATP to cAMP and another by inhibiting the hydrolysis of phosphodiester bond of cAMP to AMP.Following mitochondrial stress, the accumulated PINK1 and Parkin induces mitophagy for the removal of damaged/diseased mitochondria (15).LC3 is an ubiquitin-like protein that is covalently attached to the surface of autophagosome during its biogenesis(16).CHCHD10 S59L overexpression caused LC3 accumulation in HeLa YFP−Parkin cells as a result of active mitophagy via the PINK1/Parkin pathway.Both Forskolin and Ro umilast treatment led to a decrease in LC3 accumulation in the cells.In our Drosophila model, we also showed that Forskolin and Ro umilast reduced the C2C10H S81L -induced toxicity.
PDE4 targeted therapies have shown promising results in various neurological disorders such Alzheimer's disease (AD), Parkinson's disease (PD), Fragile X Syndrome, depression and neuropathic pain (17).At present, very few drugs are available for the treatment of ALS.Ibudilast, a PDE4 inhibitor is in different phases of clinical trials for multiple neurodgenerative diseases, including Multiple Sclerosis and ALS due to their anti-in ammatory activity.In case of ALS, Ibudilast has been shown to slow the disease progression (18,19).Apremilast is also in many clinical trials and crosses the blood brain barrier (17).Signi cantly, Apremilast was very effective in mitigating the mitochondrial fragmentation and respiration in lower doses compared to Ro umilast and Forskolin in our experiments.
Our data highlights the potential of PDE4 inhibitors as effective therapeutic agents in the context of CHCHD10 S59L -induced ALS-FTD.It also implies that the effectiveness of PDE4 inhibitors for neurodegeneration may not be the only consequence of modulating neuroin ammation as in the case of many previous clinical studies, but also regulating mitochondrial biology (which is the primary mechanism involved in CHCHD10 S59L associated ALS-FTD).It is noteworthy that TDP-43 loss increases PDE4 expression (20).It may be possible to increase the e cacy of PDE4 inhbitors against neurodegeneration by improving their ability to modulate mitochondrial biology rather than solely focusing on neuroin ammation.It will be interesting to study a synergistic effect of Forskolin and PDE4 inhibitors on the pathophysiology of ALS-FTD which might be effective in much lower doses pharmacologically and well tolerated treatment option without much side effects for this challenging neurodegenerative disorder.

CONCLUSION
In conclusion, our study demonstrates that FDA-approved PDE4 inhibitors, including Ro umilast and Apremilast, can effectively mitigate the morphological and functional mitochondrial defects induced by the CHCHD10 S59L mutation associated with ALS-FTD.On a mechanistic level, these drugs led to a reduction in the accumulation of PINK1 and Parkin on mitochondria, and subsequently decreased mitophagy, thereby preserving mitochondrial integrity and function.Our ndings suggest that currently marketed PDE4 inhibitors have the potential to be repositioned for the treatment of CHCHD10 S59Linduced ALS-FTD and potentially other related neurodegenerative disorders.This study provides a promising avenue for developing targeted therapies to modulate the PINK1/Parkin pathway in CHCHD10S59L-induced ALS-FTD.
To validate the results from Akabane et al.'s research, we tested whether an adenylate cyclase activator, Forskolin, reduces PINK1 and Parkin accumulation caused by CCCP.CCCP is a mitochondrial uncoupling agent inducing PINK1 stabilization and subsequent Parkin accumulation on mitochondria.As they demonstrated, acute Forskolin (100uM) treatment almost completely blocked Parkin accumulation induced by CCCP in HeLa cells stably expressing Parkin-YFP (Fig.

Forskolin
and Ro umilast reduce Parkin accumulation caused by CHCHD10 S59L in HeLa Parkin−YFP cells.Mitochondria-localized PINK1 phosphorylates ubiquitins and the ubiquitin-like domain of Parkin, resulting in Parkin accumulation on mitochondria.Therefore, we investigated whether Forskolin and Ro umilast reduced Parkin acculumation in HeLa cells via reducing CHCHD10 S59L -induced PINK1 accumulation.We used HeLa cells stably expressing yellow uorescent protein (YFP)-tagged Parkin.